Herpes simplex virus type 1 (HSV-1) is a medically significant pathogen. 60% of US citizens are infected and rates are higher in developing countries. Serious complications, such as permanent brain damage from encephalitis in adults or neonates, and corneal inflammation leading to blindness and poor long- term outcomes even after corneal transplantation, occur at a low per-person rate. Because infection is so prevalent, the cumulative medical impact of these complications is quite considerable. There is no licensed vaccine for HSV-1. A vaccine for HSV-2, a pathogen with overall 50% amino acid identity, had no clinical activity in a recent phase III trial. This HSV-2 vaccine targeted antibody and CD4 T-cell responses, but not CD8 T-cell responses. The long-term goal of this research is the rational design of preventative and therapeutic vaccines for HSV-1. CD8 T-cells are a critical component of the immune response to HSV-1 and localize to infected skin, cornea, retina, and trigeminal ganglia. By implication from human HSV-2 and murine HSV-1 data, CD8 cells persist at sites of healed skin infection to provide early surveillance for reactivation. Almost nothing is known about their fine specificity in humans. We have developed novel technology what allows us to study the CD8 and CD4 T-cell response to HSV-1 in unprecedented detail, including the study of peripheral blood from person who never have herpetic lesions, that is, person with immunologic success. Aim 1 uses a sequence of novel methods: efficient cross-presentation, CD137-based sorting, and a library of cloned HSV-1 ORFs, to decode the CD8 response to HSV with high throughput and definition. We will compare immune responses in persons with symptomatic and asymptomatic HSV-1 infection and identify antigens that have both high population prevalence and correlate with asymptomatic infection. HSV-1-specific CD4s localize to infected eyes, TG, and skin in humans, and to infected tissues in mice, and likely provide important direct antiviral and helper functions. Aim 2 defines with high throughput, using the complete ORFeome, the breadth, specificity, and within-subject true immunodominance hierarchy of HSV-1-specific CD4 responses. The workflow uses CD137-based sorting to and a highly antigenic HSV-1 ORFeome protein set. Dominance assays are combinatorial in two dimensions (responder cell oligoclonality and matrix ORF pooling) and deliver the fine specificity of ~100 individual HSV-1-specific CD4 T-cells per subject to define the CD4 dominance architecture. The overall goal of the Project is to identify HSV-1 ORFs for vaccines that stimulate dominant and prevalent CD8 and CD4 epitopes to persons of diverse HLA type that are correlated with mild infection.